Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit

Abstract

Cytochrome P-450 dependent oxygenase (3'-hydroxy-hexobarbital) and oxidase activities (hydrogen peroxide) have been measured in hepatic microsomes from guinea pigs, rats and rabbits. A sensitive gas-chromatographic assay was developed to measure the hydroxylated product 3'-hydroxy-hexobarbital. The kinetics of its formation were determined and correlated to hexobarbital type I binding and compared with oxidase activity: in the rat, Vmax for 3'-hydroxyhexobarbital formation was 5.1 and 2.6 nmoles/mg/min, resp. This was increased by phenobarbital treated rabbits, Vmax was 15.0 nmoles/mg/min for hydroxylation and 40.8 for H2O2 formation. Spectral affinity constants (Ks) in control animals were 0.12 mM (rats) and 0.14 mM (rabbits). Phenobarbital treatment decreased these affinity constants, which were similar for each activity measured. In guinea pigs, however, hydroxylation of exobarbital was low (3.1 nmoles/mg/min) and hexobarbital-dependent formation of H2O2 was higher than hydroxylation (Vmax: 7.0 nmoles/mg/min). Phenobarbital treatment led here to two affinity constnts for each activity measured, which however, were alike. The existence of low in addition to high affinity constants observed here might explain the difficulties seen hitherto in correlating hexobarbital binding and metabolism in this species. Total oxidase activity was higher than oxygenase activity in all species tested. It is suggested that oxygenase activity of cytochrome P-450 is not limited by binding but by a competition with oxidase activity for a common intermediary species. This might be peroxy-P-450 (substrate-Fe3+O2(2-), rendering either substrate-Fe3+ O for hydroxylation reaction, or oxidized cytochrome P-450-substrate and hydrogen peroxide as product of oxidase function.

Repression of Phenobarbital-Dependent CYP2B1 mRNA Induction by Reactive Oxygen Species in Primary Rat Hepatocyte Cultures

“…CYP2B isoforms in particular are involved in activation of aflatoxin B1 or cyclophosphamide to genotoxic metabolites (Chang et al, 1993). Furthermore, reactive oxygen species (ROS) are released during the catalytic reaction cycle of P-450 (Heinemeyer et al, 1980). It is assumed that activity of P-450 systems constitutes a major source of intracellular ROS in the liver (Bondy and Naderi, 1994;Puntarulo and Cederbaum, 1998), indicating that excessive P-450 activity resulting from P-450 induction may lead to cellular damage caused by generation of noxious metabolites or ROS.…”

The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

“…superoxide (Ingelman-Sundberg and Johansson, 1980;Kuthan et al, 1978), hydrogen peroxide (Heinemeyer et al, 1980;Oprian et al, 1983), and water (Gorsky et al, 1984;Morgan et al, 1982;Zhukov and Archakov, 1982), respectively. In in vitro reactions, hydrogen peroxide arises in two ways: by dismutation of the superoxide anion (Equation 1) and by dissociation of hydrogen peroxide from a two-electron-reduced cytochrome P-450.…”

Oxidase and Oxygenase Function of the Microsomal Cytochrome P450 Monooxygenase System

“…It was found that some substrates of the monooxygenase system stimulate Hz02 formation whereas others are either not effective or are effective only in animals pretreated with phenobarbital but not with pregnenolone-16a-carbonitrile [3,4]. A correlation of H202 formation with the spectral binding and hydroxylation of hexobarbital was observed by Heinemeyer et al [6] from which a hypothetical peroxide complex of cytochrome P450 was postulated as a common intermediate for Hz02 formation and substrate hydroxylation.If this hypothesis is correct, an increased H202 formation should be observable upon addition of pseudo-substrates [7,8] of the microsomal cytochrome P450 monooxygenase system. This was reinvestigated in the present study.As a second possible pathway of NADPH-dependent formation of H202 in liver microsomes the release of superoxide anion radicals by autoxidation of microsomal redox systems, and the subsequent disproportionation of these Saarland, Homburg-Saar, Federal Republic of Germany.…”